Parathyroid hormone (PTH) is produced in the parathyroid glands through the two-step conversion of pre-pro-PTH (115-amino acids) to pro-PTH (90-amino acids) to the 84-amino acid peptide PTH(1-84). The hormone is secreted into the circulatory system to produce basal (healthy) concentrations of ˜15-65 pg/mL, and is assayed to assist in the diagnosis of hypo/hyperparathyroidism, hypercalcemia and in monitoring for renal osteodystrophy in patients with end-stage renal failure. Conventional PTH assays typically rely on two-antibody recognition systems coupled to a variety of detection modalities (e.g., enzymatic amplification, electrochemiluminescence, and fluorescence). Notably, assays of greatest specificity are able to differentiate between different truncated forms of PTH, and are referred to as “second” or “third” generation PTH assays.
Of particular importance to these later-generation assays is their ability to selectively monitor different PTH forms of known biological consequence. Notably, two variants, full-length PTH(1-84) and PTH missing the six N-terminal amino acids (PTH(7-84)), are the subject of increased clinical investigation and potential diagnostic capability. Due to confounding microheterogeneity, these variants were historically considered as a single PTH value (i.e., by the “first” generation assays). Classification of each as its own molecular entity, and the ability to analyze and study each independently, suggests an antagonistic relationship between the two different forms in relationship to calcium homeostasis. As such, there is mounting clinical evidence that the ratio between PTH(1-84) and PTH(7-84) may differentiate between hyperparathyroid bone turnover and adynamic bone disease.
This PTH(1-84)-to-PTH(7-84) paradigm is a most recent example of describing the biological and clinical utility of microheterogeneity within the PTH protein. Indeed, other PTH variants have been defined as far back as ˜40 years ago. Perhaps the most mature of these, PTH(1-34), has been identified as an endogenous variant that exhibits biochemical activity comparable to the full-length protein. Consequently, it represents a classic example of a peptide-based bioactive variant that has transitioned through drug development to the point of FDA-approval for the treatment of osteoporosis (rPTH(1-34) (teriparatide)). Collectively the routine monitoring of these clinically-relevant PTH variants—PTH(1-84), PTH(7-84) and PTH(1-34)—is achieved through three separate, high-specificity immunometric assays. However, there are indications that even greater microheterogeneity exists within PTH, which has yet to be fully characterized to determine clinical utility and/or confounding effects on present-day assays. The accurate examination of known PTH variants, while simultaneously evaluating other possible variants, requires a degree of analytical freedom of which conventional assays are generally incapable.